Reagents for oligonucleotide cleavage and deprotection

ABSTRACT

The present invention provides a process for the removal of protecting groups, i.e. deprotection, from chemically synthesized oligonucleotides. In one embodiment, the invention provides reagents suitable for use in such a process, and kits incorporating such reagents in a convenient, ready-to-use format. By use of the process and reagents of the invention, side-reactions leading to certain impurities that contaminate the synthesized oligonucleotides can be minimized.  
     Methods and reagents are provided for deprotection of an oligonucleotide by reacting a protected oligonucleotide with a deprotection reagent wherein the deprotection reagent comprises an active methylene compound and an amine reagent. The active methylene compound has the structure:  
                 
 
     where substituent EWG is an electron-withdrawing group and R is hydrogen, C 1 -C 12  alkyl, C 6 -C 20  aryl, heterocycle or an electron-withdrawing group.

I. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. applicationSer. No. 10/091,231 filed on Mar. 4, 2002, which claims the benefitunder 35 USC §119(e) of provisional U.S. application No. 60/274,309,filed Mar. 8, 2001, which are all incorporated herein by reference.

II. FIELD OF THE INVENTION

[0002] This invention relates generally to synthetic oligonucleotidecompounds. More specifically, this invention relates to cleavage ofoligonucleotides from solid supports and deprotection ofoligonucleotides.

III. BACKGROUND OF THE INVENTION

[0003] Oligonucleotides are essential reagents in many importantmolecular biology experiments, assays and information gatheringoperations, such as the polymerase chain reaction (PCR), diagnosticprobes, single nucleotide polymorphism (SNP) detection, and genomicsequencing. The benefits of conducting the synthesis of oligonucleotidesby the sequential addition and covalent attachment of monomeric unitsonto a solid support is well appreciated. In particular, the method ofCaruthers is highly optimized and almost universally adopted (U.S. Pat.Nos. 4,458,066 and 4,973,679). The vast majority of the millions ofoligonucleotides consumed each year are prepared by automated synthesiswith phosphoramidite nucleoside monomers (Beaucage (1992) TetrahedronLett. 22:1859-62, U.S. Pat. No. 4,415,732).

[0004] Conducting chemical reactions on solid supports has severalpractical advantages: (i) excess reagents and soluble by-products can beeasily removed and separated by simple washing and filtration steps,(ii) dispensing, manipulating, organizing the parallel production ofmany oligonucleotides is facilitated, and (iii) reactions can be scaledup or down for economy and ease of handling.

[0005] Many applications utilize oligonucleotides with a covalentlyattached label. Labels may impart some function, e.g. affinity,detection, or other physical property. Oligonucleotide labels often havereactive functionality, which may preferably be protected to minimizeside reactions and modifications.

[0006] Upon completion of synthesis, the solid support-boundoligonucleotide is removed from the support by chemical cleavage of thecovalent linkage between the oligonucleotide and the solid support, anddeprotected to remove all remaining protecting groups from theoligonucleotide. The steps of cleavage and deprotection may beconcurrent and conducted with the same reagent. Alternatively, cleavageand deprotection may be conducted at different temperatures and withdifferent reagents.

[0007] Typically, cleavage of the oligonucleotide (20 μmole to 1 nmole)from the solid support is performed in the synthesis column at roomtemperature using about 1 to 3 ml concentrated ammonium hydroxide NH₄OH(about 28-30% NH₃ in water). Cleavage of the typical ester linkage atthe 3′ terminus of the oligonucleotide is complete in about one hourunder these conditions. While the linkage between the oligonucleotideand the solid support is cleaving, ammonium hydroxide is also removingthe 2-cyanoethyl groups from the internucleotide phosphates and thenucleobase protecting groups. Depending on the nucleobase and the typeof protecting groups, deprotection (removal of protecting groups) of theoligonucleotide requires approximately 1 to 8 hours at 55° C. treatmentwith concentrated ammonium hydroxide.

[0008] Alternatively, cleavage and deprotection may be conducted withanhydrous amines (U.S. Pat. No. 5,750,672), methylamine (U.S. Pat. Nos.5,348,868 and 5,518,651), hydrazine and ethanolamine (Polushin (1991)Nucleic Acids Res. Symposium Series No. 24, p. 49-50; Polushin (1994)Nucleic Acids Res. 22:639-45)

[0009] A typical post-synthesis, cleavage/deprotection routine onautomated DNA synthesizers (e.g. Models 392, 394, 3948, AppliedBiosystems, Foster City, Calif.) delivers concentrated ammoniumhydroxide through the synthesis column after completion ofoligonucleotide synthesis and allows it to stand in the column for aboutone hour, with periodic deliveries of more ammonium hydroxide andcollection of the eluant in a vessel. The vessel containing the cleavedand partially deprotected oligonucleotide can then be transferred to aheating device to complete deprotection. Alternatively, the nucleobaseprotecting groups may be sufficiently labile to not require furtherheating to yield a fully deprotected oligonucleotide. The ammoniumhydroxide is removed under vacuum or in a stream of air or inert gas.The crude oligonucleotide may be purified by various methods, includinghydrophobic cartridge purification, reverse-phase HPLC, polyacrylamidegel electrophoresis, and precipitation. For some applications, the crudeoligonucleotide may be pure enough to perform adequately.

[0010] After completion of cleavage of the oligonucleotides from thesupport, the remaining protecting groups are removed by incubation inthe ammonium hydroxide solution at either room temperature or withheating, e.g. 55° C. for 6-24 hours. Alternatively, oligonucleotides canbe cleaved and/or deprotected with ammonia, or other amines, in the gasphase whereby the reagent gas comes into contact with theoligonucleotide while attached to, or in proximity to, the solid support(U.S. Pat. Nos. 5,514,789; 5,738,829).

[0011] The particular cleavage and deprotection protocol used in anysituation is largely determined by protecting groups employed on thenucleobases, the internucleotide phosphorus, the sugars, 3′ or 5′terminus, and any covalently attached label. The first generation set ofnucleobase protecting groups utilized in the phosphodiester method ofsynthesis includes benzoyl (bz) and isobutyryl (ibu) protecting groups,utilized as adenosine A^(bz), cytosine C^(bz) and guanosine G^(ibu)(Schaller (1963) J. Amer. Chem. Soc. 85, 3821-3827 and Buchi (1972) J.Mol. Biol. 72:251). Generally, thymidine T is not protected.

[0012] It is known that certain side-reactions occur during the cleavageand deprotection reactions. Modifications of the nucleobases,internucleotide phosphate groups, and pendant amino groups have beencharacterized (Chang (1999) Nucleosides & Nucleotides 18:1205-1206;Manoharan (1999) Nucleosides & Nucleotides 18:1199-1201). Acrylonitrile,released from deprotection of the internucleotide phosphate groups, mayform adducts on the nucleobases, labels, or other sites (EP 1028124; WO0046231; Eritja (1 992) Tetrahedron 48:4171-82; Wilk (1999) J. Org.Chem. 64:7515-22). Other impurities are uncharacterized, but known todetract from the purity of oligonucleotides and cause loss ofperformance. Where deprotection of protecting groups is incomplete,oligonucleotides may hybridize with lower specificity or affinity,leading to mispriming or mutagenicity.

[0013] New reagents and methods for cleavage and deprotection ofoligonucleotides are desirable. Certain protecting groups may not becompatible with deprotection reagents or automated synthesizers andprotocols, leading to modifications. Certain labels, e.g. those withextended conjugation or reactive functionality, may lead tomodifications of the labels or the oligonucleotide during the cleavageand deprotection steps. Reagents and methods which minimize or eliminateside reactions and modifications are desirable.

IV. SUMMARY

[0014] The present invention provides a process for the removal ofprotecting groups, i.e. deprotection, from chemically synthesizedoligonucleotides. In one embodiment, the invention provides reagentssuitable for use in such a process, and kits incorporating such reagentsin a convenient, ready-to-use format. By use of the process and reagentsof the invention, side-reactions leading to certain impurities thatcontaminate the synthesized oligonucleotides can be minimized.

[0015] In a first aspect, the invention provides a method fordeprotection of an oligonucleotide by reacting a protectedoligonucleotide with a deprotection reagent wherein the deprotectionreagent comprises an active methylene compound and an amine reagent. Theactive methylene compound has the structure:

[0016] The substituent EWG is an electron-withdrawing group selectedfrom nitro, ketone, ester, carboxylic acid, nitrile, sulfone, sulfonate,sulfoxide, phosphate, phosphonate, nitroxide, nitroso, trifluoromethyland aryl groups substituted with one or more nitro, ketone, ester,carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide, phosphate,phosphonate, nitroxide, nitroso, and trifluoromethyl. The substituent Ris selected from hydrogen, C₁-C₁₂ alkyl, C₆-C₂₀ aryl, heterocycle andelectron-withdrawing group. The amine reagent may be aqueous ammoniumhydroxide, aqueous methylamine, or anhydrous C₁-C₆ alkylamine. Inaddition to an active methylene compound and an amine reagent, thedeprotection reagent of the invention may include water or an alcoholsolvent. Protecting groups are removed from the oligonucleotide bytreatment with the deprotection reagent.

[0017] The oligonucleotide may be covalently attached to a solid supportthrough a linkage. The oligonucleotide may be cleaved from the solidsupport either before, during, or after the protecting groups areremoved. The solid support may be an organic polymer or inorganic. Thesolid support may be a membrane or frit which allows the deprotectionreagent to pass through.

[0018] The solid support may be confined in a column or other enclosurewhich has inlet and outlet openings for the deprotection reagents topass or flow through. The columns may be configured in a variety offormats, including holders of many columns, e.g. 96- or 384-wellmicrotitre plate formats. A plurality of oligonucleotides in a holdermay be deprotected concurrently or separately through discriminate orindiscriminate delivery or exposure to the deprotection reagents.

[0019] Oligonucleotides which may be deprotected by the deprotectionreagents of the invention include nucleic acid analogs. Oligonucleotidesmay bear one or more covalently attached labels such as a fluorescentdye, a quencher, biotin, a mobility-modifier, and a minor groove binder.

[0020] In a second aspect, the invention provides a method fordeprotection of an oligonucleotide by first wetting the protectedoligonucleotide covalently attached to the solid support with an activemethylene compound and a solvent, and then reacting the protectedoligonucleotide with an amine reagent. The amine reagent may be inliquid or gas phase; aqueous or anhydrous, e.g. aqueous ammoniumhydroxide, ammonia gas or a C₁-C₆ alkylamine.

[0021] In a third aspect, the invention includes an oligonucleotidedeprotection reagent wherein the deprotection reagent comprises anactive methylene compound and an amine reagent. The active methylenecompound has the structure:

[0022] The substituent EWG is an electron-withdrawing group selectedfrom nitro, ketone, ester, carboxylic acid, nitrile, sulfone, sulfonate,sulfoxide, phosphate, phosphonate, nitroxide, nitroso, trifluoromethyland aryl groups substituted with one or more nitro, ketone, ester,carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide, phosphate,phosphonate, nitroxide, nitroso, and trifluoromethyl. The substituent Ris selected from hydrogen, C₁-C₁₂ alkyl, C₆-C₂₀ aryl, heterocycle andelectron-withdrawing group. The active methylene compound may be 1 to10% by volume of the deprotection reagent. The deprotection reagent mayfurther include an alcohol solvent which is 1 to 30% by volume of thereagent.

[0023] In a fourth aspect, the invention includes deprotectedoligonucleotides deprotected by the deprotection reagents of theinvention.

V. BRIEF DESCRIPTION OF THE FIGURES

[0024]FIGS. 1a-1 b show reverse-phase HPLC chromatograms of T₁₅-Q-CDPI₃,cleaved and deprotected with 15% ethanol:NH₄OH only (FIG. 1a) and with3% diethylmalonate (DEM) in 15% ethanol:NH₄OH (FIG. 1b).

[0025]FIGS. 2a-2 d show reverse-phase HPLC chromatograms of 5′ F-CAG TCGCCC TGC C-Q-CDPI₃ 3′ (SEQ ID. NO 3) cleaved and deprotected with 15%ethanol:NH4OH and either 0% DEM (FIG. 2a), 0.1% DEM (FIG. 2b), 1% DEM(FIG. 2c), or 3% DEM (FIG. 2d).

[0026]FIGS. 3a-3 d show reverse-phase HPLC chromatograms of 5′ F-CTT CTTGCT AAT TCC-Q-CDPI₃ 3′ (SEQ ID. NO 4) cleaved and deprotected with 15%ethanol:NH4OH and either 0% DEM (FIG. 3a), 0.1% DEM (FIG. 3b), 1% DEM(FIG. 3c), or 3% DEM (FIG. 3d).

[0027]FIGS. 4a-4 b show reverse-phase HPLC chromatograms of 5° F-CCA TGCGTT AGC C-Q-CDPI₃ 3′ (SEQ ID. NO. 5) cleaved and deprotected with 15%ethanol:NH₄OH only (FIG. 4a) and with 3% diethylmalonate (DEM) in 15%ethanol:NH₄OH (FIG. 4b).

[0028]FIGS. 5a-5 b show reverse-phase HPLC chromatograms of 5′H₂N-(PEO)₂-AAA ATC AAG AAC TAC AAG ACC GCC C 3′ (SEQ ID. NO. 6) cleavedand deprotected with concentrated NH4OH only (FIG. 5a) and with 1%diethylmalonate (DEM) in 15% ethanol:NH₄OH (FIG. 5b).

VI. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0029] Reference will now be made in detail to certain embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with theillustrated embodiments, it will be understood that they are notintended to limit the invention to those embodiments. On the contrary,the invention is intended to cover all alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

[0030] VI.1 Definitions

[0031] Unless stated otherwise, the following terms and phrases as usedherein are intended to have the following meanings:

[0032] “Nucleobase” means a nitrogen-containing heterocyclic moietycapable of forming Watson-Crick hydrogen bonds in pairing with acomplementary nucleobase or nucleobase analog, e.g. a purine, a7-deazapurine, or a pyrimidine. Typical nucleobases are the naturallyoccurring nucleobases adenine, guanine, cytosine, uracil, thymine, andanalogs of the naturally occurring nucleobases, e.g. 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine (U.S. Pat.No. 5,912,340), inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isoguanine,2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil,O⁶-methylguanine, N⁶-methyladenine, O⁴-methylthymine,5,6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, phenoxazine,7-deazapurine, pseudo-isocytidine, isoguanosine, 4(3H)-pyrimidone,hypoxanthine, 8-oxopurines, pyrazolo[3,4-D]pyrimidines (U.S. Pat. Nos.6,143,877 and 6,127,121) and ethenoadenine (Fasman (1989) PracticalHandbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press,Boca Raton, Fla.).

[0033] “Nucleoside” means a compound consisting of a nucleobase linkedto the C-1′ carbon of a ribose sugar. The ribose may be substituted orunsubstituted. Substituted ribose sugars include, but are not limitedto, those riboses in which one or more of the carbon atoms, e.g., the2′-carbon atom, is substituted with one or more of the same or different—R, —OR, —NRR or halogen groups, where each R is independently hydrogen,C₁-C₆ alkyl or C₅-C₁₄ aryl. Sugars include ribose, 2′-deoxyribose,2′,3′-dideoxyribose, 2′-haloribose, 2′-fluororibose, 2′-chlororibose,2′-C-alkyl, 2′-alkylribose, e.g. 2′-O-methyl, 4′-α-anomeric nucleotides,1′-α-anomeric nucleotides, 2′-4′- and 3′-4′-linked and other “locked”,bicyclic sugar modifications (WO 98/22489; WO 98/39352; WO 99/14226).Modifications at the 2′- or 3′-position include hydrogen, hydroxy,methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl,alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo.When the nucleobase is purine, e.g. A or G, the ribose sugar is attachedto the N⁹-position of the nucleobase. When the nucleobase is pyrimidine,e.g. C, T or U, the pentose sugar is attached to the N¹-position of thenucleobase (Kornberg and Baker, (1992) DNA Replication, 2^(nd) Ed.,Freeman, San Francisco, Calif.).

[0034] “Nucleotide” means a phosphate ester of a nucleoside, as amonomer unit or within a nucleic acid. Nucleotides are sometimes denotedas “NTP”, or “dNTP” and “ddNTP” to particularly point out the structuralfeatures of the ribose sugar. “Nucleotide 5′-triphosphate” refers to anucleotide with a triphosphate ester group at the 5′ position. Thetriphosphate ester group may include sulfur substitutions for thevarious oxygens, e.g. α-thio-nucleotide 5′-triphosphates.

[0035] As used herein, the terms “oligonucleotide” and “polynucleotide”are used interchangeably and mean single-stranded and double-strandedpolymers of nucleotide monomers, including 2′-deoxyribonucleotides (DNA)and ribonucleotides (RNA) linked by internucleotide phosphodiester bondlinkages, or internucleotide analogs, and associated counter ions, e.g.,H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺and the like. A polynucleotide maybe composed entirely of deoxyribonucleotides, entirely ofribonucleotides, or chimeric mixtures thereof. Polynucleotides may becomprised of internucleotide, nucleobase and sugar analogs.Polynucleotides typically range in size from a few monomeric units, e.g.5-40, when they are frequently referred to as oligonucleotides, toseveral thousand monomeric nucleotide units. Unless denoted otherwise,whenever a polynucleotide sequence is represented, it will be understoodthat the nucleotides are in 5′ to 3′ order from left to right and that“A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, unless otherwise noted.

[0036] “Protected oligonucleotide” means any oligonucleotide orpolynucleotide prepared by synthesis means, e.g. phosphoramiditenucleoside method of automated synthesis on solid support, whichincludes one or more protecting groups on functional groups such as theexocyclic amine of a nucleobase, the internucleotide phosphate linkage,or 5′ terminus hydroxyl or amine. Protecting group terminology followsthe general strategies taught by Greene, T. and Wuts, P. “ProtectiveGroups in Organic Synthesis”, Third Edition, John Wiley & Sons, Inc.,New York, N.Y. (1999).

[0037] The term “nucleic acid analogs” refers to analogs of nucleicacids comprising one or more nucleotide analog units, and possessingsome of the qualities and properties associated with nucleic acids, e.g.Watson/Crick, wobble, and Hoogsteen base pairing, and other sequencerecognition effects. Nucleic acid analogs may have modified nucleobasemoieties, modified sugar moieties, and/or modified internucleotidelinkages (Englisch (1991) Angew. Chem. Int. Ed. Engl. 30:613-29).Modifications include labels. One class of nucleic acid analogs is wherethe internucleotide moiety is modified to be neutral and uncharged at ornear neutral pH, such as phosphoramidate, phosphotriester, and methylphosphonate oligonucleotides where one of the non-bridging oxygen atomsis replaced by a neutral substituent, e.g. —NR₂, —OR, —R. Another classof nucleic acid analogs is where the sugar and internucleotide moietieshave been replaced with an uncharged, neutral amide backbone, such asmorpholino-carbamate and peptide nucleic acids (PNA). A form of PNA is aN-(2-aminoethyl)-glycine amide backbone polymer (Nielsen, 1991).Whenever a PNA sequence is represented, it is understood that the aminoterminus is at the left side and the carboxyl terminus is at the rightside.

[0038] “Deprotection reagent” means any reagent or formulation in aliquid or gaseous state which removes a protecting group from aprotected oligonucleotide by chemical reaction, or cleaves anoligonucleotide from a solid support.

[0039] “Solid support” means any particle, bead, or surface upon whichsynthesis of an oligonucleotide occurs.

[0040] “Active methylene compound” means any organic reagent which bearsan acidic proton bound to carbon and capable of removal under basicconditions, typically with a pKa of about 6 to 20.

[0041] The terms “cleaving” or “cleavage” refer to breaking a covalentbond that attaches an oligonucleotide to a solid support.

[0042] The term “label”, as used herein, means any moiety which can beattached to an oligonucleotide and that functions to: (i) provide adetectable signal; (ii) interact with a second label to modify thedetectable signal provided by the first or second label, e.g. FRET;(iii) stabilize hybridization, i.e. duplex formation; (iv) affectmobility, e.g. electrophoretic mobility or cell-permeability, by charge,hydrophobicity, shape, or other physical parameters, or (v) provide acapture moiety, e.g., affinity, antibody/antigen, or ionic complexation.

[0043] The terms “linker”, “LINKER”, and “linkage” are usedinterchangeably and mean a chemical moiety comprising a covalent bond ora chain of atoms that covalently attaches, or is attached to, a label toa polynucleotide, one label to another, or a solid support to apolynucleotide or nucleotide.

[0044] “Linking moiety” means a chemically reactive group, substituentor moiety, e.g. a nucleophile or electrophile, capable of reacting withanother molecule to form a covalent bond, or linkage.

[0045] “Substituted” as used herein refers to a molecule wherein one ormore hydrogen atoms are replaced with one or more non-hydrogen atoms,functional groups or moieties. For example, an unsubstituted nitrogen is—NH₂, while a substituted nitrogen is —NHCH₃. Exemplary substituentsinclude but are not limited to halo, e.g., fluorine and chlorine,(C₁-C₈) alkyl, sulfate, sulfonate, sulfone, amino, ammonium, amido,nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic,heterocycle, water-solubilizing group, and linking moiety. “Alkyl” meansa saturated or unsaturated, branched, straight-chain, branched, orcyclic hydrocarbon radical derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkane, alkene, or alkyne. Typicalalkyl groups consist of 1-12 saturated and/or unsaturated carbons,including, but not limited to, methyl, ethyl, propyl, butyl, and thelike.

[0046] “Alkoxy” means —OR where R is (C₁-C₆) alkyl.

[0047] “Alkyldiyl” means a saturated or unsaturated, branched, straightchain or cyclic hydrocarbon radical of 1-20 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkane, alkeneor alkyne. Typical alkyldiyl radicals include, but are not limited to,1,2-ethyldiyl, 1,3-propyldiyl, 1,4-butyldiyl, and the like.

[0048] “Aryl” means a monovalent aromatic hydrocarbon radical of 6-20carbon atoms derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene,substituted benzene, naphthalene, anthracene, biphenyl, and the like.

[0049] “Aryldiyl” means an unsaturated cyclic or polycyclic hydrocarbonradical of 6-20 carbon atoms having a conjugated resonance electronsystem and at least two monovalent radical centers derived by theremoval of two hydrogen atoms from two different carbon atoms of aparent aryl compound.

[0050] “Heterocycle” means any ring system having at least onenon-carbon atom in a ring.

[0051] “Substituted alkyl”, “substituted alkyldiyl”, “substituted aryl”and “substituted aryldiyl” mean alkyl, alkyldiyl, aryl and aryldiylrespectively, in which one or more hydrogen atoms are each independentlyreplaced with another substituent. Typical substituents include, but arenot limited to, —X, —R, —OH, —OR, —SR, —SH, —NH₂, —NHR, —NR₂, —⁺NR₃,—N═NR₂, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, —N₂ ⁺, —N₃,—NHC(O)R, —C(O)R, —C(O)NR₂—S(O)₂O⁻, —S(O)₂R, —OS(O)₂OR, —S(O)₂NR,—S(O)R, —OP(O)(OR)₂, —P(O)(OR)₂, —P(O)(O⁻)₂, —P(O)(OH)₂, —C(O)R, —C(O)X,—C(S)R, —C(O)OR, —CO₂ ⁺, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NR₂, —C(S)NR₂,—C(NR)NR₂, where each X is independently a halogen and each R isindependently —H, C₁-C₆ alkyl, C₅-C₁₄ aryl, heterocycle, or linkinggroup.

[0052] “Intemucleotide analog” means a phosphate ester analog of anoligonucleotide such as: (i) alkylphosphonate, e.g. C₁-C₄alkylphosphonate, especially methylphosphonate; (ii) phosphoramidate;(iii) alkylphosphotriester, e.g. C₁-C₄ alkylphosphotriester; (iv)phosphorothioate; and (v) phosphorodithioate. Internucleotide analogsalso include non-phosphate analogs wherein the sugar/phosphate subunitis replaced by an a non-phosphate containing backbone structure. Onetype of non-phosphate oligonucleotide analogs has an amide linkage, suchas a 2-aminoethylglycine unit, commonly referred to as PNA (Nielsen(1991) “Sequence-selective recognition of DNA by strand displacementwith a thymidine-substituted polyamide”, Science 254:1497-1500).

[0053] “Water solubilizing group” means a substituent which increasesthe solubility of the compounds of the invention in aqueous solution.Exemplary water-solubilizing groups include but are not limited toquaternary amine, sulfate, sulfonate, carboxylate, phosphonate,phosphate, polyether, polyhydroxyl, and boronate.

[0054] “Array” means a predetermined spatial arrangement ofoligonucleotides present on a solid support or in an arrangement ofvessels.

[0055] VI.2 Oligonucleotide Synthesis

[0056] Oligonucleotides which are cleaved and deprotected by thereagents and methods of the invention may be synthesized on solidsupports by the phosphoramidite method (U.S. Pat. Nos. 4,415,732 and4,973,679; Beaucage, S. and Iyer, R. (1992) Tetrahedron 48:2223-2311)using: (1) 3′ phosphoramidite nucleosides, I (2) supports e.g. silica,controlled-pore-glass (U.S. Pat. No. 4,458,066) and polystyrene (U.S.Pat. Nos. 5,047,524 and 5,262,530), and (3) automated synthesizers(Models 392, 394, 3948, 3900 DNA/RNA Synthesizers, Applied Biosystems).Other support materials include polyacrylate, hydroxethylmethacrylate,polyamide, polyethylene, polyethyleneoxy, or copolymers and grafts ofsuch.

[0057] Generally, the phosphoramidite method of synthesis is preferredbecause of efficient and rapid coupling and the stability of thestarting nucleoside monomers. The method entails cyclical addition ofmonomers, e.g. structure I, to an oligonucleotide chain growing on asolid-support, most commonly in the 3′ to 5′ direction in which the 3′terminus nucleoside is attached to the solid-support at the beginning ofsynthesis through a linkage. The linkage typically includes base-labilefunctionality, such as a succinate, diglycolate, oxalate, orhydroquinone-diacetate (Pon (1997) Nucleic Acids Res. 25:3629-35) and iscleavable by ammonia, amines, carbonate, hydroxide, and other basicreagents. The 3′ phosphoramidite nucleoside monomer units arecommercially available and share the general structure I:

[0058] where, R₁ is a protecting group or substituent, e.g.2-cyanoethyl, methyl, lower alkyl, substituted alkyl, phenyl, aryl, andsubstituted aryl; R₂ and R₃ are amine substituents, e.g. isopropyl,morpholino, methyl, ethyl, lower alkyl, cycloalkyl, and aryl; R₄ is anexocyclic nitrogen protecting group such as benzoyl, isobutyryl, acetyl,phenoxyacetyl, aryloxyacetyl, phthaloyl (U.S. Pat. No. 5,936,077),2-(4-nitro-phenyl)ethyl, pent-4-enoyl, dimethylformamidine (dmf),dialkylformamidine, and dialkylacetamidine; and R₅ is an acid-labileprotecting group such as 4, 4′-dimethoxytrityl (DMT), 4-methoxytrityl(MMT), pixyl, trityl, and trialkylsilyl. Alternatively, oligonucleotidescan be synthesized in the 5′ to 3′ direction with 5′ phosphoramiditenucleoside monomers, e.g. the 5′ bears a phosphoramidite group and the3′ bears an acid-labile protecting group (Wagner (1997) Nucleosides &Nucleotides, 16:1657-60).

[0059] Cleavage and deprotection with the reagents and methods of theinvention may be conducted on oligonucleotides with more labile linkagesto a solid support and more labile protecting groups. More labilenucleobase protecting groups are commercially available, e.g.,phenoxyacetyl type: Expedite™ (Sinha (1993) Biochimie 75:13-23;available from Applied Biosystems, Foster City, Calif.) and PAC™phosphoramidites (U.S. Pat. No. 4,980,460; Schulhof (1987) Nucleic AcidsRes. 15:397-416; Schulhof (1988) Nucleic Acids Res. 16:319; availablefrom Amersham Pharmacia), and formamidines and acetamidines (McBride(1986) J. Amer. Chem. Soc. 108:2040-48; Froehler (1983) Nucleic AcidsRes. 11:8031-36; Theisen (1993) Nucleosides & Nucleotides 12:1033-46).These labile protecting groups are deprotected significantly faster thanthe first generation set. For example, the set A^(bz), C^(bz), G^(dmf),T (Fastphoramidite™, Applied Biosystems, Foster City, Calif.) requiresonly one hour at 65° C. in concentrated ammonium hydroxide for completedeprotection.

[0060] The invention may be practiced on oligonucleotides which arecovalently attached to any solid support through a linkage. The solidsupport may be any material, in any configuration, dimension, or scaleupon which the oligonucleotide may be attached or synthesized. Typicalsolid supports include beads or particles of highly cross-linkedpolystyrene (U.S. Pat. Nos. 5,047,524; 5,262,530) orcontrolled-pore-glass. Dimensionally, solid supports may beapproximately 1 to 100 μm average diameter and monodisperse or widelyvariant in size and shape. The beads or particles may be enclosed in acolumn having inlet and outlet openings. Reagents for conducting thephosphoramidite method of synthesis may be made to flow through a columnmounted on the automated synthesizer. Alternatively, the solid supportmay be a porous membrane, filter, frit, or other flow-through device orconfiguration which conducts similar reagent flow.

[0061] Alternatively, the solid support may be an impermeable, rigidorganic polymer, such as polyvinylchloride, polyethylene, polystyrene,polyacrylate, polycarbonate and copolymers thereof. Yet another solidsupport may be a non-porous, planar material such as glass, quartz, ordiamond (EP 1063286). Suitable materials also include metals, e.g.aluminum, gold, platinum, silver, copper, and the like, or alloysthereof. The metals may be solid blocks, or surfaces, including layers.The materials may have at least one substantially planar surface in aslide, sheet, plate, or disc configuration (WO 01/01142). In oneembodiment, a block material such as glass is coated with a metalliclayer or thin film such as gold, silver, copper or platinum. Depositionof metal films may be conducted by methods such as electron beamevaporation. The metallic layer is derivatized with reactivefunctionality to which is attached an oligonucleotide. For example, agold layer may be derivatized with a disulfide linkage to the 3′ or 5′terminus of an oligonucleotide.

[0062] Inorganic solid supports such as glass, controlled-pore-glass,silica gel are typically derivatized with silane reagents such asaminoalkyl-trialkoxysilanes or mercaptoalkyl-trialkoxysilanes, whichyield amino and thiol functional groups, respectively. Oligonucleotides,the initial nucleoside for oligonucleotide synthesis, or universalsupport reagents may then be covalently attached to the amino or thiolderivatized solid supports.

[0063] An array of solid support surfaces upon which oligonucleotidesmay be synthesized or attached may be made to undergo cleavage ordeprotection with the reagents, and by the methods of the invention, ina parallel or sequential fashion. One or a subset of the protectedoligonucleotides on an array may be selectively cleaved and deprotectedby masking, targeted delivery of reagents, or other means of directingexposure to the reagents (Fodor, U.S. Pat. No. 5,445,934).

[0064] VI.3 Methods of Oligonucleotide Cleavage and Deprotection

[0065] Upon completion of synthesis, the solid support-boundoligonucleotide is removed from the support by chemical cleavage of thecovalent linkage between the oligonucleotide and the solid support, anddeprotected to remove all remaining protecting groups from theoligonucleotide, including P from nucleobases and cyanoethyl from theinternucleotide linkages. The steps of cleavage and deprotection may becoincidental and conducted with the same reagent, e.g. concentratedammonium hydroxide when P is an amide type protecting group and LINKERis an ester, structure II. Alternatively, the steps of cleavage anddeprotection can be conducted separately with “orthogonal” reagents. Forexample, when LINKER is disulfide, the nucleobase P and phosphateprotecting groups may be removed from a protected oligonucleotide withammonium hydroxide and the deprotected oligonucleotide will remainattached to the solid support. Conversely, the same protectedoligonucleotide may be cleaved from the solid support with itsprotecting groups intact with a disulfide-selective cleaving reagent,such as dithiothreitol. The net result of cleavage and deprotection isexemplified by the structures of a protected oligonucleotide II and acleaved and deprotected oligonucleotide III:

[0066] In one embodiment of the invention, the 3′ terminus of aprotected oligonucleotide is represented by structure II, showing 2nucleotides of 5 to about 100 nucleotides. The nucleobases are protectedwith base-labile protecting groups, P. An exemplary set is A^(bz),G^(ibu), C^(bz), and T. The internucleotide phosphate groups may beprotected by 2-cyanoethyl, methyl, or some other protecting group. The3′ terminus is attached through a linkage, LINKER, to a solid support,S, in structure II. The linkage includes base-labile functionality suchas ester, carbamate, or phosphate (EP 839 829). Typically the 3′ esteris succinate, diglycolate, oxalate, or hydroquinone-diacetate. Aftersynthesis, the protected oligonucleotide is reacted with a deprotectionreagent of the invention to effect removal of nucleobase protectinggroups, P, and internucleotide phosphate protecting groups,2-cyanoethyl. Concurrently or separately, the 3′ terminus linkage iscleaved to separate the oligonucleotide from the solid support toultimately yield the cleaved and deprotected oligonucleotide shown bystructure III.

[0067] In another embodiment, the linkage is chosen to be non-cleaving,i.e. resistant to cleavage during synthesis and deprotection steps. Anon-cleaving linkage may contain inert types of functionality such asamide, alkyl, phosphate, or ether functionality. An oligonucleotidesynthesized with a non-cleaving linkage may be deprotected by thereagents and methods of the invention and utilized in a solid-phaseformat, e.g. a biochip, DNA chip, or array, where a plurality ofdeprotected oligonucleotides are immobilized on a solid substrate. Agrid or matrix of solid-support bound oligonucleotides may be thusarrayed in known locations and addressable by complementary nucleicacids or other reagents, light, a laser, current, or detectionapparatus.

[0068] In another embodiment, the linkage to a solid support is chosento be selectively cleavable, i.e. resistant to cleavage during synthesisand deprotection steps but cleavable with other reagents or conditions.A linkage to a solid support may be selectively cleavable when itcontains a C—Si or an O—Si bond and cleavage is conducted with afluoride anion reagent, e.g. tetra-butylammonium fluoride ortriethylammonium hydrogen fluoride. A linkage may be selectivelycleavable when it contains disulfide, —S—S—, functionality and iscleaved by dithiothreitol or other disulfide cleaving reagents. Alinkage may be selectively cleavable when it contains anortho-nitrobenzyl group and is cleaved under photolysis conditions.

[0069] A surprising and unexpected aspect of the invention is that adeprotection reagent including an active methylene compound and an aminereagent is effective and efficient at cleavage and deprotection ofoligonucleotides. The novel deprotection reagents and methods of theinvention may minimize undesired side reactions leading to impurities ormodifications of oligonucleotides, including their covalently attachedlabels. The amine reagent serves as a nucleophile to displace theprotecting groups and the active methylene compound serves to react withor render inert certain intermediates which may further react to modifythe oligonucleotide or any label on the oligonucleotide. Othermechanisms may occur and other benefits may accrue from use of thedeprotection reagent of the invention.

[0070] In one embodiment, the amine reagent and active methylenecompound are mixed together to provide a deprotection reagent that canbe applied to a protected oligonucleotide to remove protecting groups(Examples 1-3). The reaction may be conducted at room temperature or atan elevated temperature. When the protected oligonucleotide iscovalently attached to a solid support through a linkage, the process ofremoving protecting groups may be concurrent with cleaving theoligonucleotide from the solid support. After cleavage, the cleavedoligonucleotide may be separated from the solid support by filtrationthrough a frit or membrane, or by decantation. The cleavedoligonucleotide may be further deprotected under an elevated temperatureor with addition of other reagents to assist in the removal ofprotecting groups. When deprotection is complete, the deprotectedoligonucleotide may be separated from the deprotection reagents byconventional, well-known means such as evaporation, precipitation,electrophoresis, chromatography, or hydrophobic cartridge procedures.One or more of the compounds in the deprotection reagent may besufficiently volatile to be removed by evaporation under a stream of gasor under vacuum.

[0071] The amine reagent may be used in a liquid formulation or in agaseous state (Boal (1996) Nucleic Acids Res. 24:3115-17). Certainamines are gases at room temperature and pressure, such as ammonia(bp=−33° C.) and are effective at removing oligonucleotide protectinggroups and conducting cleavage (Kempe, U.S. Pat. No. 5,514,789). Theprotected oligonucleotide may be contacted by ammonia gas in anenclosed, pressurized space, container, or bomb. The ammonia may bedelivered through a conduit from a pressurized vessel as a gas (Kempe,U.S. Pat. No. 5,738,829), or generated from aqueous ammonium hydroxidewithin an enclosed space that also includes the protectedoligonucleotide. In the latter embodiment, ammonia gas or a vapor ofammonia and water may be created by enclosure in an enclosed space of anopen container, e.g. a pan or flask, of ammonium hydroxide solution. Thegaseous state of the reagent increases in concentration by raising thetemperature in the enclosed space (Example 6).

[0072] In another embodiment, the active methylene compound may contactthe protected oligonucleotide prior to the amine reagent, or in amixture including the amine reagent. In one embodiment, the activemethylene compound and a solvent are mixed and used to wet a solidsupport to which a protected oligonucleotide is covalently attached. Asufficient volume of the mixture is delivered to cover the solid supportor wet the surface, e.g. in a flow-through vessel such as a column(Example 6). The amine reagent is delivered next to the solid support,ensuring that a sufficient amount of the active methylene reagent isretained. The side-reaction suppression benefits of the active methylenereagent is thus realized by a sequential delivery of reagents.

[0073] VI.4 Reagents For Oligonucleotide Cleavage and Deprotection

[0074] Oligonucleotides may be cleaved and/or deprotected by novelreagents of the invention which include an active methylene compound andan amine reagent. Active methylene compounds include organic reagentswhich bear an acidic proton bound to carbon capable of removal underbasic conditions, typically with a pKa of about 6 to 20. The activemethylene compound may constitute 1 to 10% by volume of the deprotectionreagent. Active methylene compounds are represented by the structure:

[0075] where the acidity of the carbon group is increased by anelectron-withdrawing group (EWG). Other substituents (R) on the acidiccarbon may be a second or third electron-withdrawing group, hydrogen,alkyl, aryl, or any functional group which renders the proton acidic inthe range of about pKa=6-20. Electron-withdrawing groups include nitro,ketone, ester, carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide,phosphate, phosphonate, nitroxide, nitroso, and trifluoromethyl.Electron-withdrawing groups also include aryl groups substituted withone or more nitro, ketone, ester, carboxylic acid, nitrile, sulfone,sulfonate, sulfoxide, phosphate, phosphonate, nitroxide, nitroso, andtrifluoromethyl groups. Useful classes of active methylene compoundsinclude: (i) 1,3 keto-esters, e.g. ethylacetoacetate; (ii) 1,3diketones, e.g. 2,4-pentanedione and cyclohexanedione, (iii) malonatederivatives, e.g. malononitrile, malonic acid, malonamide, anddialkylmalonate diesters. Dialkylmalonate diesters includedimethylmalonate, diethylmalonate (DEM), di-n-propylmalonate, anddiisopropylmalonate.

[0076] The effects of the concentration of an active methylene compoundwere investigated with four ethanolic ammonia (15% ethanol:conc. NH₄OH)reagents containing 0%, 0.1%, 1%, and 3% of diethylmalonate (FIGS. 2a-2d). Each of the four reagents was used to cleave and deprotect a portionof an oligonucleotide labelled with a fluorescent dye, a quenchermoiety, and a minor groove binder (Example 3). Analysis by reverse phaseHPLC showed significant contaminating impurities in the reagent withoutan active methylene compound (0% DEM). The presence of 0.1% DEMeliminated most of the impurities. The presence of 1% and 3% essentiallyeliminated all late eluting impurities.

[0077] In an embodiment where the active methylene compound is dissolvedin a solvent and used to wet the solid support to which a protectedoligonucleotide is covalently attached, prior to treatment with theamine reagent, the solvent may be selected from an alcohol, an ether, anamide, acetonitrile, dichloromethane, or dimethylsulfoxide. Alcoholsolvents include methanol, ethanol, n-propanol, isopropanol, or1,2-ethylene glycol. Ether solvents include diethyl ether,tetrahydrofuran, 1,4-dioxane, or 1,2-dimethoxyethane. Amide solventsinclude acetamide, formamide, benzamide, or dimethylformamide

[0078] The amine reagent may be used in the gaseous state or dissolvedin water, as a solution to treat the oligonucleotide on the solidsupport. The composition of the amine reagent includes any reagent witha primary, secondary, or tertiary amino group which reacts with aprotected oligonucleotide to effect removal of the protecting groups.Amine reagents thus include: (i) ammonia (NH₃) gas; (ii) ammoniadissolved as ammonium hydroxide (NH₄OH) in water or mixtures of waterand alcohol solvents; (iii) alkylamines, R₂NH and RNH₂ where R is C₁-C₆alkyl; (iv) alkyl and aryldiamines, H₂N—R—NH₂, where R is C₁-C₂₀alkyldiyl or C₆-C₂₀ aryldiyl; and (v) formamidines such as1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and1,5-diazabicyclo[4.3.0]non-5-ene (DBN).

[0079] Alcohol solvents include methanol, ethanol, ethylene glycol,isopropanol, and other hydroxyl containing reagents which assist insolubilizing the reagents, wetting the solid support, increasingreaction rates, or minimizing side-reactions. The alcohol solvent mayconstitute 1 to 30% by volume of the deprotection reagent.

[0080] The amine reagent may contact the oligonucleotide in the gaseousstate, generated from a solution in a closed system or environment withthe oligonucleotide. For example, the protected oligonucleotide bound toa solid support may be enclosed in a container which further contains anopen vessel of ammonium hydroxide solution. The container may be sealed,or open to the atmosphere. When sealed, the container may be heated, inthe manner of a bomb apparatus. The ammonia vapors may thus contact theoligonucleotide and remove protecting groups. Alternatively, the aminereagent may be passed through, or delivered to, a column or vesselcontaining the oligonucleotide. For example, the amine reagent may beinstalled on an automated synthesizer and delivered to a column, as partof the programmed delivery of reagents which may flow through the inletand outlet openings of the column. The active methylene reagent may bedelivered to the vessel containing the oligonucleotide prior to theamine reagent, or as a mixture with the amine reagent.

[0081] One or more columns in which the oligonucleotides are synthesizedmay be placed in, or transferred to, a holder apparatus, e.g. microtitrewell tray, in which the method of deprotection of the invention may beconducted. The holder may be enclosed in a sealable vessel in whichdeprotection reagents are also placed or delivered. For example, aholder containing protected oligonucleotides on solid supports incolumns can be sealed in a stainless steel pressure vessel. Adeprotection reagent can either be placed in the vessel before sealing,or delivered through a conduit into the vessel. In this general manner,a plurality, e.g. several or hundreds, of oligonucleotides may besimultaneously cleaved and deprotected. Alternatively, more than oneholder of columns may be sealed in the vessel. Also, the holders may beintroduced and processed serially, by manual intervention, or programmedrobotic means.

[0082] VI.5 Labelled Oligonucleotides

[0083] Oligonucleotides to be cleaved and deprotected by the novelreagents and methods of the invention may be conjugated, “labelled” withlabel reagents. Such conjugates may find utility as DNA sequencingprimers, PCR primers, oligonucleotide hybridization probes,oligonucleotide ligation probes, double-labelled 5′-exonuclease(TaqMan™) probes, size standards for electrophoresis, i.e. “lanestandards” or “lane markers”, and the like (U.S. Pat. No. 4,757,141;Andrus, “Chemical methods for 5′ non-isotopic labelling of PCR probesand primers” (1995) in PCR 2: A Practical Approach, Oxford UniversityPress, Oxford, pp. 39-54; Hermanson, Bioconjugate Techniques, (1996)Academic Press, San Diego, Calif. pp. 40-55, 643-71; Mullah (1998) Nucl.Acids Res. 26:1026-1031).

[0084] Certain labels provide a signal for detection of the labelledoligonucleotide by fluorescence, chemiluminescence, or electrochemicalluminescence (Kricka, L. in Nonisotopic DNA Probe Techniques (1992),Academic Press, San Diego, pp. 3-28). Fluorescent dyes useful forlabelling oligonucleotides include fluoresceins, rhodamines (U.S. Pat.Nos. 5,366,860; 5,847,162; 5,936,087; 6,008,379; 6,191,278),energy-transfer dyes (U.S. Pat. Nos. 5,863,727; 5,800,996; 5,945,526),and cyanines (Kubista, WO 97/45539). Examples of fluorescein dyesinclude 6-carboxyfluorescein; 2′,4′,1,4,-tetrachlorofluorescein; and2′,4′,5′,7′,1,4-hexachlorofluorescein (Menchen, U.S. Pat. No.5,118,934). Fluorescence has largely supplanted radioactivity as thepreferred detection method for many ligation experiments andapplications, such as the oligonucleotide ligation assay and other invitro DNA probe-based diagnostic tests.

[0085] Another class of labels includes fluorescence quenchers. Theemission spectra of a quencher overlaps with a proximal intramolecularor intermolecular fluorescent dye such that the fluorescence of thefluorescent dye is substantially diminished, or quenched, by thephenomenon of fluorescence resonance energy transfer “FRET” (Clegg(1992) “Fluorescence resonance energy transfer and nucleic acids”, Meth.Enzymol. 211:353-388). An example of FRET in the present invention iswhere the oligonucleotide is labelled with a fluorescent dye and afluorescence quencher. Particular quenchers include but are not limitedto (i) rhodamine dyes selected from the group consisting oftetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine(ROX); (ii) diazo compounds, e.g. DABSYL, DABCYL (Matayoshi (1990)Science 247:954-58; Tyagi, WO 95/13399), Fast Black, (Nardone, U.S. Pat.No. 6,117,986); (iii) cyanine dyes (Lee, U.S. Pat. No. 6,080,868) and,(iv) other chromophores e.g. anthraquinone, malachite green,nitrothiazole,and nitroimidazole compounds and the like.

[0086] Energy-transfer dyes are another type of oligonucleotide label.An energy-transfer dye label includes a donor dye linked to an acceptordye (U.S. Pat. No. 5,800,996). Light, e.g. from a laser, at a firstwavelength is absorbed by a donor dye, e.g. FAM. The donor dye emitsexcitation energy absorbed by the acceptor dye. The acceptor dyefluoresces at a second, longer wavelength. The donor dye and acceptordye moieties of an energy-transfer label may be attached by a linkagelinking the 4′ or 5′ positions of the donor dye, e.g. FAM, and a 5- or6-carboxyl group of the acceptor dye. Other rigid and non-rigid linkagesmay be useful.

[0087] Metal porphyrin complexes, e.g. aluminum phthalocyaninetetrasulfonate (Stanton, WO 88/04777) and chemiluminescent compounds.e.g 1,2-dioxetane chemiluminescent moieties (Bronstein, U.S. Pat. No.4,931,223) are other examples of useful oligonucleotide labels.

[0088] Another class of labels, referred to herein ashybridization-stabilizing moieties, include but are not limited to minorgroove binders (Blackburn, M. and Gait, M. Nucleic Acids in Chemistryand Biology (1996) Oxford University Press, p.337-46), intercalators,polycations, such as poly-lysine and spermine, and cross-linkingfunctional groups. Hybridization-stabilizing moieties may increase thestability of base-pairing, i.e. affinity, or the rate of hybridization,exemplified by high thermal melting temperatures, Tm, of the duplex.Hybridization-stabilizing moieties may also increase the specificity ofbase-pairing, exemplified by large differences in Tm between perfectlycomplementary oligonucleotide and target sequences and where theresulting duplex contains one or more mismatches of Watson/Crickbase-pairing (Blackburn, M. and Gait, M. Nucleic Acids in Chemistry andBiology (1996) Oxford University Press, pp. 15-81). Labels which enhancehybridization specificity and affinity are desirable, e.g. minor-groovebinders and affinity ligand labels. Biotin and digoxigenin are usefulaffinity ligand labels for the capture and isolation ofoligonucleotides. Minor groove binders include Hoechst 33258, CDPI₁₋₃(U.S. Pat. No. 6,084,102; WO 96/32496; Kutyavin (2000) Nucleic AcidsRes. 28:655-61), netropsin, and distamycin. Other useful labels includeelectrophoretic mobility modifiers, amino acids, peptides, and enzymes.

[0089] A labelled oligonucleotide may have formula IV:

[0090] where the oligonucleotide comprises 2 to 1000 nucleotides. LABELis a protected or unprotected form of a fluorescent dye, an exemplaryclass of labels, which includes an energy-transfer dye. B is anynucleobase, e.g. uracil, thymine, cytosine, adenine, 7-deazaadenine,guanine, and 8-deazaguanosine. L is a linkage, such as a propargyl amine(U.S. Pat. Nos. 5,047,519; 5,770,716; 5,821,356; 5,948,648). R⁶ is H,OH, halide, azide, amine, C₁-C₆ aminoalkyl, C₁-C₆ alkyl, allyl,protected hydroxyl, trialkylsilyloxy, tert-butyldimethylsilyloxy,C₁-C₆alkoxy, OCH₃, or OCH₂CH═CH₂. R⁷ is H, phosphate, internucleotidephosphodiester, or internucleotide analog. R⁸ is H, phosphate,internucleotide phosphodiester, or internucleotide analog. In thisembodiment, the nucleobase-labelled oligonucleotide IV may bear multiplelabels attached through the nucleobases. Nucleobase-labelledoligonucleotide IV may be formed by: (i) coupling of a nucleosidephosphoramidite reagent by automated synthesis or (ii) post-synthesiscoupling with a label reagent. Oligonucleotides labelled at the 5′terminus have structure V:

[0091] where X is O, NH, or S; R⁶ is H, OH, halide, azide, amine, C₁-C₆aminoalkyl, C₁-C₆ alkyl, allyl, C₁-C₆ alkoxy, —OCH₃, or —OCH₂CH═CH₂; R⁷is H, phosphate, internucleotide phosphodiester, or internucleotideanalog; and L is C₁-C₁₂ alkyldiyl, C₆-C₂₀ aryldiyl, or polyethyleneoxyof up to 100 ethyleneoxy units.

[0092] A variety of labels may be covalently attached at the 3′ terminusof oligonucleotides. A solid support bearing a label, or bearingfunctionality which can be labelled by a post-synthesis reaction, can beutilized as a solid support for oligonucleotide synthesis (U.S. Pat.Nos. 5,141,813; 5,231,191, 5,401,837; 5,736,626). By this approach, thelabel or the functionality is present during synthesis of theoligonucleotide. During cleavage and deprotection, the label or thefunctionality remains covalently attached to the oligonucleotide.Oligonucleotides labelled at the 3′ terminus may have structure VI:

[0093] The linkage L in formulas IV, V, VI may be attached at any siteon the label, LABEL.

[0094] Labelling can be accomplished using any one of a large number ofknown techniques employing known labels, linkages, linking groups,standard reagents and reaction conditions, and analysis and purificationmethods. Generally, the linkage linking the label and theoligonucleotide should not (i) interfere with hybridization, (ii)inhibit enzymatic activity, or (iii) adversely affect the properties ofthe label, e.g. quenching or bleaching fluorescence of a dye.Oligonucleotides can be labelled at sites including a nucleobase, asugar, an internucleotide linkage, and the 5′ and 3′ terminii.Oligonucleotides can be functionalized to bear reactive amino, thiol,sulfide, disulfide, hydroxyl, and carboxyl groups at any of these sites.Nucleobase label sites generally include the 7-deaza or C-8 positions ofthe purine or deazapurine, and the C-5 position of the pyrimidine. Thelinkage between the label and the nucleobase may be acetylenic-amido oralkenic-amido linkages. Typically, a carboxyl group on the label isactivated by forming an active ester, e.g. N-hydroxysuccinimide (NHS)ester and reacted with an amino group on the alkynylamino- oralkenylamino-derivatized nucleobase. Labels are most conveniently andefficiently introduced at the 5′ terminus (Andrus, A. “Chemical methodsfor 5′ non-isotopic labelling of PCR probes and primers” (1995) in PCR2: A Practical Approach, Oxford University Press, Oxford, pp. 39-54)with fluorescent dyes and other labels which have been functionalized asphosphoramidite reagents, as part of the automated protocol.

[0095] Oligonucleotides may be labelled at both the 5′ and 3′ terminii.Each terminii may bear one or more labels. For example, Examples 1-4include oligonucleotides with a 5′ fluorescent dye and two labels, aquencher Q and a minor groove binder CDPI₃, at the 3′ terminus.

[0096] In a first method for labelling synthetic oligonucleotides, anucleophilic functionality, e.g. a primary aliphatic amine, isintroduced at a labelling attachment site on an oligonucleotide, e.g. a5′ terminus. After automated, solid-support synthesis is complete, theoligonucleotide is cleaved from the support and all protecting groupsare removed. The nucleophile-oligonucleotide is reacted with an excessof a label reagent containing an electrophilic moiety, e.g.isothiocyanate or activated ester, e.g. N-hydroxysuccinimide (NHS),under homogeneous solution conditions (Hermanson, BioconjugateTechniques, (1996) Academic Press, San Diego, Calif. pp. 40-55, 643-71;Andrus, A. “Chemical methods for 5′ non-isotopic labelling of PCR probesand primers” (1995) in PCR 2: A Practical Approach, Oxford UniversityPress, Oxford, pp. 39-54). Labelled oligonucleotides IV, V, or VI may beformed by reacting a reactive linking group form, e.g. NHS, of a dye,with an oligonucleotide functionalized with an amino, thiol, or othernucleophile (U.S. Pat. No. 4,757,141).

[0097] In a second method, a label is directly incorporated into theoligonucleotide during or prior to automated synthesis, for example as asupport reagent (U.S. Pat. Nos. 5,736,626 and 5,141,813) or as anon-nucleoside phosphoramidite reagent. Certain fluorescent dyes andother labels have been functionalized as phosphoramidite reagents for 5′labelling (Theisen (1992) Nucleic Acid Symposium SeriesNo. 27, OxfordUniversity Press, Oxford, pp. 99-100).

[0098] Polynucleotides may be labelled with moieties that affect therate of electrophoretic migration, i.e. mobility-modifying labels.Mobility-modifying labels include polyethyleneoxy units, —(CH₂CH₂O)_(n)—where n may be 1 to 100 (U.S. Pat. No. 5,624,800). The polyethyleneoxyunits may be interspersed with phosphate groups. Specifically labellingpolynucleotides with labels of polyethyleneoxy of discrete and knownsize allows for separation by electrophoresis, substantially independentof the number of nucleotides in the polynucleotide. That is,polynucleotides of the same length may be discriminated upon by thepresence of spectrally resolvable dye labels and mobility-modifyinglabels. Polynucleotides bearing both dye labels and mobility-modifyinglabels may be formed enzymatically by ligation or polymerase extensionof the single-labelled polynucleotide or nucleotide constituents.

[0099] The present invention is particularly well suited for cleavingand deprotecting polynucleotides with multiple and different labels.

VI.6 EXAMPLES

[0100] The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention and not to in any way limit its scope.

Example 1

[0101] An oligonucleotide T₈-Q-CDPI₃: 5′ TTT TTT TT-Q-CDPI₃ 3′ (SEQ ID.NO. 1)

[0102] was synthesized on the Model 3948 DNA Synthesizer (AppliedBiosystems, Foster City, Calif.). Eight cycles of phosphoramiditechemistry was conducted with thymidine 3′ phosphoramidite in a columncontaining 16 mg (200 nmoles) highly cross-linked polystyrene beadsupport loaded with 12 μmole/gm of a linkage including quencher label Qand minor groove binder label CDPI₃. The quencher label, Q, has thestructure:

[0103] where X is the attachment site to a linkage. Theminor-groove-binder label, CDPI₃, has the following structure:

[0104] where X is the attachment site to a linkage.

[0105] The support was divided into two portions. The first portion wastreated with 15% ethanolic ammonia (15:85 v/v EtOH:conc. NH₄OH) for 2hours at 55° C. to effect cleavage and deprotection. The second portionwas treated with 3% diethylmalonate (DEM) dissolved in 15% ethanolicammonia (3:15:82 v/v/v DEM:EtOH:conc. NH4OH), for 2 hours at 55° C.

[0106] After cooling, an aliquot from each portion was analyzed byreverse phase HPLC. The adsorbent was 2-5 μm particles of C-18polystyrene/divinylbenzene. The mobile phases were a gradient ofacetonitrile in TEAA (triethylammonium acetate) at about pH 7.(Transgenomic WAVE, Transgenomic, Inc., San Jose, Calif.). Other mobilephases, conditions, and HPLC equipment are also useful for analyzing theoligonucleotides which are cleaved and deprotected by the methods andreagents of the invention. The major, product peak and the major (first)late-eluting contaminant were separated and isolated from each aliquot.The late-eluting contaminant(s) from the first portion, cleaved anddeprotected without DEM, were analyzed by MALDI-TOF mass spectrometry(PerSeptive Biosystems Voyager-DE, Framingham, Mass.) and found to havea mass of 3485.5 [M+26] mass units. This mass is consistent with anadditional vinyl group modification (—CH₂═CH₂). The major peak in theHPLC from each portion was assigned to T₈-Q-CDPI₃ from the strongmolecular ion peak at 3459.41 mass units (positive mode), as expected.

Example 2

[0107] An oligonucleotide T₁₅-Q-CDPI₃: 5′ TTT TTT TTT TTT TTT-Q-CDPI₃ 3′(SEQ ID. NO. 2)

[0108] was synthesized on the Model 3948 DNA Synthesizer (AppliedBiosystems, Foster City, Calif.). Fifteen cycles of phosphoramiditechemistry was conducted with thymidine 3′ phosphoramidite in a columncontaining 16 mg (200 nmoles) highly cross-linked polystyrene beadsupport loaded with 12 μmole/gm of a linkage including quencher label Qand minor groove binder label CDPI₃. The support was divided into twoportions. The first portion was treated with 15% ethanolic ammonia(15:85 v/v EtOH:conc. NH4OH) for 2 hours at 55° C. to effect cleavageand deprotection. The second portion was treated with 3% diethylmalonate(DEM) dissolved in 15% ethanolic ammonia (3:15:82 v/v/v DEM:EtOH:conc.NH₄OH), for 2 hours at 55° C. After cooling, an aliquot from eachportion was analyzed by reverse phase HPLC. The portion cleaved anddeprotected without DEM shows a complex-product mixture containing only26.5% of the desired product eluting at 6.1 minutes (FIG. 1a). Theproduct mixture is contaminated with significant (50%) later elutingimpurities. The portion cleaved and deprotected with 3% DEM showsimproved purity, 76.8% of the desired product eluting at 6.1 minutes anda diminished level of later eluting impurities (FIG. 1b).

EXAMPLE 3

[0109] Oligonucleotides labelled with a fluorescent dye(F=6-carboxyfluorescein) at the 5′ terminus, and a quencher moiety (Q)and minor groove binder (CDPI₃) at the 3′ terminus: 5′ F-CAG TCG CCC TGCC-Q-CDPI₃ 3′ (SEQ ID. NO. 3) 5′ F-CTT CTT GCT AAT TCC-Q- (SEQ ID. NO. 4)CDPI₃ 3′

[0110] were synthesized on a Model 3900 DNA Synthesizer (AppliedBiosystems, Foster City, Calif.). Phosphoramidite chemistry wasconducted with nucleoside 3′ phosphoramidites, including A^(bz),G^(dmf), C^(bz) and T, in a column containing 16 mg (200 nmoles) highlycross-linked polystyrene bead support loaded with 12 μmole/gm of alinkage including quencher label Q and minor groove binder label CDPI₃.

[0111] After each synthesis, the support was divided into four portions.Each portion was treated with a reagent containing 0%, 0.1%, 1% or 3%diethylmalonate (DEM) in 15% ethanolic ammonia (15:85 v/v EtOH:conc.NH₄OH) for 2 hours at 65° C. to effect cleavage and deprotection.

[0112] After cooling, an aliquot from each portion was analyzed byreverse phase HPLC. The portions cleaved and deprotected without DEMshows a complex product mixture containing only 21.8% of the desiredproduct eluting at 6.5 minutes (FIG. 2a) and 32.7% of the desiredproduct eluting at 6.4 minutes (FIG. 3a) for SEQ ID. NO 3 and SEQ ID. NO4 respectively. The product mixtures are contaminated with significant(50%) later eluting impurities. The portions cleaved and deprotectedwith 0.1% DEM show improved purities; 65.8% (FIG. 2b) and 64.4% (FIG.3b) and diminished levels of later eluting impurities for SEQ ID. NO 3and SEQ ID. NO 4 respectively. The portions cleaved and deprotected with1% DEM show again improved purities; 76.7% (FIG. 2c) and 76.7% (FIG. 3c)for SEQ ID. NO 3 and SEQ ID. NO 4 respectively. The portions cleaved anddeprotected with 3% DEM show again improved purities, 79.5% (FIG. 2d)and 77.5% (FIG. 3d) for SEQ ID. NO 3 and SEQ ID. NO 4 respectively.

[0113] The fluorescent dye, 6-carboxyfluorescein, (F) has the followingstructure:

[0114] where X is the attachment site to a linkage.

Example 4

[0115] Following the procedures of Example 3, the 13 nt oligonucleotide:

5′ F-CCA TGC GTT AGC C -Q-CDPI₃ 3  (SEQ ID. NO. 5)

[0116] was synthesized and the support was divided into two portions.One portion was cleaved and deprotected with 15% ethanol:NH₄OH only andwith 3% DEM in 15% ethanol:NH₄OH. An aliquot from each portion wasanalyzed by reverse phase HPLC. The portion cleaved and deprotectedwithout DEM shows a complex product mixture containing only 26% of thedesired product eluting at 6.1 minutes (FIG. 4a). The product mixture iscontaminated with significant later eluting impurities. The portioncleaved and deprotected with 3% DEM shows improved purity, 67% of thedesired product eluting at 6.1 minutes and a diminished level of latereluting impurities (FIG. 4b).

Example 5

[0117] Liquid Phase Cleavage/deprotection:

[0118] A set of up to 48 oligonucleotides are synthesized on the Model3948 DNA Synthesizer (Applied Biosystems, Foster City, Calif.). Eacholigonucleotide is synthesized at 50-100 nmolar scale on about 20 mg of3′ nucleoside, high-crosslink polystyrene in a OneStep™synthesis/purification column (Applied Biosystems, Foster City, Calif.;Andrus, U.S. Pat. Nos. 5,935,527 and 6,175,006; Baier (1996)BioTechniques 20:298-303). Oligonucleotides may be 15-50 nt, or longer.Oligonucleotides may be unlabelled or labelled with labels such asfluorescent dyes or hybridization-stabilizing moieties. Synthesis isconducted with the FastPhoramidite™ set of 3′ phosphoramiditenucleosides (A^(bz), G^(dmf), C^(bz), T) dissolved in acetonitrile andcoupled to the 5′ terminus of the growing oligonucleotide withtetrazole, or a tetrazole analog, e.g. 5-ethylthiotetrazole, as aproton-source activator. Synthesis may be programmed to either removethe 5′ DMT group from the 5′ terminus of the oligonucleotide by acidicdetritylation, or leave it intact by omitting the final detritylationstep. When a set of three oligonucleotides finishes the synthesis stageunder the synthesis fluid delivery head, the set of three columnsrotates under the cleavage/deprotection delivery head. The deprotectionreagent of the invention may be delivered to the columns, e.g. 0.5 to1.5 ml of a mixture of concentrated ammonium hydroxide and an activemethylene compound. The active methylene compound may be 1 to 10% byvolume of the reagent. The deprotection reagent may further contain 1 to30% of an alcohol solvent, by volume. The deprotection reagent isallowed to stand in, or circulate through, the column at ambient orhigher temperature for several minutes to an hour. The oligonucleotideis thereby cleaved from the solid support and can be delivered toenclosed tubing which is heated at about 65° C. for about 1-2 hours tocomplete deprotection, i.e. removal of nucleobase and internucleotideprotecting groups.

[0119] When the 5′ DMT group has been left intact, the solutioncontaining the deprotected oligonucleotide may be purified bytrityl-selective hydrophobic interaction by absorption onto thepolystyrene in the OneStep column in which it was synthesized. Followingabsorption (loading), the column is treated with reagents to effectwashing away of impurities, detritylation of the oligonucleotide, andelution of the deprotected, purified, and detritylated oligonucleotide.

Example 6

[0120] Gas Phase Cleavage/deprotection:

[0121] A set of 48 oligonucleotides were synthesized in a singlepre-programmed run on the Model 3900 DNA Synthesizer (AppliedBiosystems, Foster City, Calif.). Each oligonucleotide was synthesizedat a 200 nmolar scale on about 10 mg of polystyrene support in a columnwith inlet and outlet openings. The oligonucleotides ranged in size from15 to 30 nt in length. After synthesis of the 48 oligonucleotides wascomplete, 200 μl of a 1% DEM in acetonitrile solution was delivered toeach column. Argon gas was flushed through the openings for about 30seconds to expel most of the solution. The columns were then transferredto a holder, e.g. 96 well microtiter format. The holder was placed in asealable stainless steel, pressure vessel with an internal volume ofapproximately one gallon. Up to four such holders could be placed in thevessel for parallel cleavage and deprotection operations. The holderswere placed on a mesh screen affixed approximately 1 inch from thebottom of the vessel. Approximately 450 ml of chilled, concentratedammonium hydroxide solution was added to the bottom floor of the vessel,or into a shallow pan that sits on the bottom floor of the vessel, belowthe mesh screen. The columns or holders were not in direct contact withthe ammonium hydroxide solution. The vessel was sealed and heated to 65°C. for about 2 hours. The pressure generated inside during the heatingperiod was about 45 psi. The vessel was cooled, vented, and opened.

[0122] The holders containing the columns were removed from the vesseland placed in a device whereby a vacuum can be applied to draw liquidsand air through the inlet opening of the columns. To each column, 250 μlof water was delivered and pulled through to waste. The cleaved anddeprotected oligonucleotides were eluted by delivering 250 μl of 20%(50% for labelled oligonucleotides) acetonitrile in water to each columnand collecting the eluant in a vessel mounted below the outlet openingof the column. Alternatively, the liquid reagents, i.e. water wash oreluant solution, can be drawn through the column by centrifugation wherethe holder is rotated in a centrifuge. The eluted oligonucleotides canbe dried under vacuum and resuspended in an aqueous medium, furtherdiluted, or used directly by aliquot in experiments.

Example 7

[0123] Following the procedures of Example 3, the 25 nt oligonucleotide:

5′ H₂N—(PEO)₂— AAA ATC AAG AAC TAC AAG ACC GCC C3′   (SEQ ID. NO. 6)

[0124] was synthesized on C polystyrene support. After the final Aphosphoramidite was coupled, two PEO (pentaethyleneoxy; —(CH₂CH₂O)₅—)linkers were coupled as PEO phosphoramidite, followed by Aminolink TFA(Applied Biosystems, Foster City, Calif.) phosphoramidite to give the 5′amino with 2 PEO linkages (Vinayak, WO 00/50432; Andrus, WO 98/39353).The support was divided into two portions. One portion was cleaved anddeprotected with NH₄OH only. The other portion was cleaved andeprotected with 1% DEM in 15% ethanol:NH₄OH. An aliquot from eachportion was analyzed by reverse phase HPLC. The portion cleaved anddeprotected with NH4OH only shows a complex product mixture containingonly 25.8% of the desired product eluting at 6.5 minutes (FIG. 5a). Theproduct mixture is contaminated with significant later elutingimpurities. The portion cleaved and deprotected with 1% DEM showsimproved purity, 48.9% of the desired product eluting at 6.5 minutes anddiminished levels of earlier and later eluting impurities (FIG. 5b).

[0125] All publications, patents, and patent applications referred toherein are hereby incorporated by reference, and to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

[0126] Although only a few embodiments have been described in detailabove, those having ordinary skill in the chemical arts will clearlyunderstand that many modifications are possible in these embodimentswithout departing from the teachings thereof. All such modifications areintended to be encompassed within the scope of the following claims.

1 6 1 8 DNA Unknown Synthetic DNA 1 tttttttt 8 2 15 DNA UnknownSynthetic DNA 2 tttttttttt ttttt 15 3 13 DNA Unknown Synthetic DNA 3cagtcgccct gcc 13 4 15 DNA Unknown Synthetic DNA 4 cttcttgcta attcc 15 513 DNA Unknown Synthetic DNA 5 ccatgcgtta gcc 13 6 25 DNA UnknownSynthetic DNA 6 aaaatcaaga actacaagac cgccc 25

We claim:
 1. A method for deprotection of an oligonucleotide comprisingthe step of reacting a protected oligonucleotide with a deprotectionreagent wherein the deprotection reagent comprises an active methylenecompound and an amine reagent, wherein the active methylene compound hasthe structure:

where EWG is an electron-withdrawing group selected from nitro, ketone,ester, carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide,phosphate, phosphonate, nitroxide, nitroso, trifluoromethyl and arylgroups substituted with one or more nitro, ketone, ester, carboxylicacid, nitrile, sulfone, sulfonate, sulfoxide, phosphate, phosphonate,nitroxide, nitroso, and trifluoromethyl; and R is selected fromhydrogen, C₁-C₁₂ alkyl, C₆-C₂₀ aryl, heterocycle andelectron-withdrawing group; whereby protecting groups are removed fromthe oligonucleotide.
 2. The method of claim 1 wherein the protectedoligonucleotide is covalently attached to a solid support through alinkage.
 3. The method of claim 2 further comprising the step ofcleaving the oligonucleotide from the solid support.
 4. The method ofclaim 2 wherein the oligonucleotide remains covalently attached to thesolid support after reacting with the deprotection reagent.
 5. Themethod of claim 2 wherein the solid support comprises highlycross-linked polystyrene.
 6. The method of claim 2 wherein the solidsupport comprises controlled-pore-glass.
 7. The method of claim 2wherein the solid support is a membrane which allows the deprotectionreagent to pass through.
 8. The method of claim 2 wherein the solidsupport is a frit which allows the deprotection reagent to pass through.9. The method of claim 2 wherein the solid support is a planar,non-porous material.
 10. The method of claim 9 wherein the material isglass, quartz, or diamond.
 11. The method of claim 9 wherein thematerial is polystyrene, polyethylene, polypropylene, nylon, graft ofpolystyrene and polyethylene glycol, copolymer of ethylene and acrylate,or copolymer of ethylene and methacrylate.
 12. The method of claim 2wherein the solid support is positioned in a column having inlet andoutlet openings whereby reagents may flow through the column.
 13. Themethod of claim 12 further comprising placing a plurality of suchcolumns in a holder and concurrently deprotecting a plurality ofoligonucleotides.
 14. The method of claim 13 wherein the holder is amicrotiter plate having an array of such columns.
 15. The method ofclaim 1 wherein the protected oligonucleotide comprises at least one2-cyanoethyl phosphate internucleotide linkage.
 16. The method of claim1 wherein the protected oligonucleotide comprises a nucleic acid analog.17. The method of claim 16 wherein the nucleic acid analog is LNA. 18.The method of claim 16 wherein the nucleic acid analog is PNA.
 19. Themethod of claim 16 wherein the nucleic acid analog is 2′-O-methyl RNA.20. The method of claim 1 wherein the protected oligonucleotide iscovalently attached to a label.
 21. The method of claim 20 wherein thelabel is selected from the group consisting of a fluorescent dye, aquencher, biotin, a mobility-modifier, a minor groove binder, and alinker selected from C₁-C₆ alkylamine and C₁-C₆ alkylthiol.
 22. Themethod of claim 21 wherein the minor groove binder is CDPI-3.
 23. Themethod of claim 21 wherein the fluorescent dye is a fluorescein, arhodamine, or a cyanine dye.
 24. The method of claim 20 wherein thelabel is attached to the 5′-terminus of the polynucleotide.
 25. Themethod of claim 20 wherein the label is attached to the 3′-terminus ofthe polynucleotide.
 26. The method of claim 1 wherein the deprotectionreagent further comprises water.
 27. The method of claim 1 wherein thedeprotection reagent further comprises an alcohol solvent.
 28. Themethod of claim 27 wherein the alcohol solvent is methanol.
 29. Themethod of claim 27 wherein the alcohol solvent is ethanol.
 30. Themethod of claim 27 wherein the alcohol solvent is ethylene glycol. 31.The method of claim 1 wherein the active methylene compound is2,4-pentanedione.
 32. The method of claim 1 wherein the active methylenecompound is 1,3-cyclohexanedione.
 33. The method of claim 1 wherein theactive methylene compound is ethyl acetoacetate.
 34. The method of claim1 wherein the active methylene compound is malononitrile
 35. The methodof claim 1 wherein the active methylene compound is malonic acid. 36.The method of claim 1 wherein the active methylene compound isnitromethane.
 37. The method of claim 1 wherein the active methylenecompound is malonamide.
 38. The method of claim 1 wherein the activemethylene compound is a dialkylmalonate diester wherein the alkyl groupsare C₁-C₆ alkyl.
 39. The method of claim 1 wherein the deprotectionreagent comprises a mixture of a dialkylmalonate diester wherein thealkyl groups are C₁-C₆ alkyl, aqueous ammonium hydroxide, and an alcoholsolvent.
 40. The method of claim 39 wherein the dialkylmalonate diesteris dimethylmalonate.
 41. The method of claim 39 wherein thedialkylmalonate diester is diethylmalonate.
 42. The method of claim 39wherein the dialkylmalonate diester is di-n-propylmalonate.
 43. Themethod of claim 39 wherein the dialkylmalonate diester isdiisopropylmalonate.
 44. The method of claim 1 wherein the amine reagentis aqueous ammonium hydroxide.
 45. The method of claim 1 wherein theamine reagent is aqueous methylamine.
 46. The method of claim 1 whereinthe amine reagent is ethylamine.
 47. The method of claim 1 wherein theamine reagent is isopropylamine.
 48. The method of claim 1 wherein theamine reagent is n-propylamine.
 49. The method of claim 1 wherein theamine reagent is n-butylamine.
 50. The method of claim 1 wherein theamine reagent is 1,2-ethylenediamine.
 51. The method of claim 1 whereinthe amine reagent is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or1,5-diazabicyclo[4.3.0]non-5-ene (DBN).
 52. The method of claim 2wherein said reacting step is effected by: wetting the protectedoligonucleotide covalently attached to the solid support with an activemethylene compound and a solvent, and then treating the protectedoligonucleotide with an amine reagent.
 53. The method of claim 52wherein the solid support is confined in a column having inlet andoutlet openings whereby reagents may flow through the column.
 54. Themethod of claim 53 wherein a plurality of columns are configured in aholder whereby a plurality of oligonucleotides are deprotectedconcurrently.
 55. The method of claim 55 wherein the holder is in amicrotiter well configuration of equally spaced columns.
 56. The methodof claim 52 further comprising the step wherein the protectedoligonucleotide and the amine reagent are placed in a sealable vesselwhereby the oligonucleotide is deprotected.
 57. The method of claim 52wherein the amine reagent is aqueous ammonium hydroxide.
 58. The methodof claim 52 wherein the amine reagent is ammonia gas.
 59. The method ofclaim 52 wherein the amine reagent is a C₁-C₆ alkylamine.
 60. The methodof claim 52 wherein the solvent is an alcohol, an ether, an amide,acetonitrile, dichloromethane, or dimethylsulfoxide.
 61. The method ofclaim 60 wherein the alcohol is methanol, ethanol, n-propanol,isopropanol, or 1,2-ethylene glycol.
 62. The method of claim 60 whereinthe ether is diethyl ether, tetrahydrofuran, 1,4-dioxane, or1,2-dimethoxyethane.
 63. The method of claim 60 wherein the amide isacetamide, formamide, benzamide, or dimethylformamide.
 64. Anoligonucleotide deprotection reagent comprising an active methylenecompound and an amine reagent wherein the active methylene compound hasthe structure

where EWG is an electron-withdrawing group selected from nitro, ketone,ester, carboxylic acid, nitrile, sulfone, sulfonate, sulfoxide,phosphate, phosphonate, nitroxide, nitroso, trifluoromethyl and arylgroups substituted with one or more nitro, ketone, ester, carboxylicacid, nitrile, sulfone, sulfonate, sulfoxide, phosphate, phosphonate,nitroxide, nitroso, and trifluoromethyl; and R is hydrogen, C₁-C₁₂alkyl, C₆-C₂₀ aryl, heterocycle or electron-withdrawing group.
 65. Theoligonucleotide deprotection reagent of claim 64 wherein the activemethylene compound is a dialkylmalonate diester and the amine reagent isaqueous ammonium hydroxide.
 66. The oligonucleotide deprotection reagentof claim 65 wherein the dialkylmalonate diester is dimethylmalonate. 67.The oligonucleotide deprotection reagent of claim 65 wherein thedialkylmalonate diester is diethylmalonate.
 68. The oligonucleotidedeprotection reagent of claim 65 wherein the dialkylmalonate diester isdi-n-propylmalonate.
 69. The oligonucleotide deprotection reagent ofclaim 65 wherein the dialkylmalonate diester is diisopropylmalonate. 70.The oligonucleotide deprotection reagent of claim 65 wherein the activemethylene compound is 1 to 10% by volume of the reagent.
 71. Theoligonucleotide deprotection reagent of claim 65 further comprising analcohol solvent.
 72. The oligonucleotide deprotection reagent of claim71 wherein the alcohol solvent is 1 to 30% by volume of the reagent. 73.A deprotected oligonucleotide deprotected by the deprotection reagent ofclaim
 64. 74. The deprotected oligonucleotide of claim 73 wherein thedeprotected oligonucleotide comprises a nucleic acid analog.
 75. Thedeprotected oligonucleotide of claim 74 wherein the nucleic acid analogis LNA.
 76. The deprotected oligonucleotide of claim 74 wherein thenucleic acid analog is PNA.
 77. The deprotected oligonucleotide of claim74 wherein the nucleic acid analog is 2′-O-methyl RNA.
 78. Thedeprotected oligonucleotide of claim 73 wherein the deprotectedoligonucleotide is covalently attached to a label.
 79. The deprotectedoligonucleotide of claim 78 wherein the label is selected from afluorescent dye, a quencher, biotin, a mobility-modifier, a minor groovebinder, and a linker selected from C₁-C₆ alkylamine and C₁-C₆alkylthiol.
 80. The deprotected oligonucleotide of claim 79 wherein theminor groove binder is CDPI-3.
 81. The deprotected oligonucleotide ofclaim 79 wherein the fluorescent dye is a fluorescein, a rhodamine, or acyanine dye.
 82. The deprotected oligonucleotide of claim 78 wherein thelabel is attached to the 5′-terminus of the polynucleotide.
 83. Thedeprotected oligonucleotide of claim 78 wherein the label is attached tothe 3′-terminus of the polynucleotide.